Hypoxia is known to affect both endothelial cell adenine nucleotide metabolism and release of vasoactive substances, but the link between these is unknown. Since the release of these vasoactive substances may be in part mediated by an increase in [Ca2+]i we investigated the effects of metabolic inhibitors on [Ca2+]i in indo-1 loaded cultured rat aortic endothelial cells. Inhibition of oxidative phosphorylation (NaCN, 2 mM) or substrate deprivation alone (no glucose) caused little change in [Ca2+]i. In contrast, combined inhibition of oxidative phosphorylation and substrate deprivation (NaCN, no glucose) caused a significant increase in [Ca2+]i. [Ca2+]i rose similarly even when cells were studied in the absence of external Ca2+. The greatest increase in [Ca2+]i occurred in the absence of substrate and during exposure to an inhibitor of glycolysis (iodoacetate, 2 mM). These results suggest a lack of endogenous oxidative substrate and a critical dependence on glycolytic metabolism.